Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C245/00—Compounds containing chains of at least two nitrogen atoms with at least one nitrogen-to-nitrogen multiple bond
  • C07C245/22—Compounds containing chains of at least two nitrogen atoms with at least one nitrogen-to-nitrogen multiple bond containing chains of three or more nitrogen atoms with one or more nitrogen-to-nitrogen double bonds
  • C07C245/24—Chains of only three nitrogen atoms, e.g. diazoamines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2601/00—Systems containing only non-condensed rings
  • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
  • C07C2601/14—The ring being saturated

Definitions

• the present invention relates to the use of bistriazene compounds as chemotherapeutic agents useful in the treatment of various cancers. As such, these compounds find wide utility in both human and veterinary medicine.
• the invention also relates to the use of these compounds as crosslinking reagents useful in a wide variety of laboratory and chemical applications involving the synthesis and manipulation of polymeric macromolecules.
• chemotherapeutic agents which act as alkylating agents capable of forming covalent linkages with a variety of substances, including phosphate groups in DNA. Alkylation of bases in DNA often leads to gene miscoding, serious damage to the DNA molecule, and/or major disruption in nucleic acid function, and results in the inhibition of a wide range of other cellular functions. These agents act by forming lethal crosslinks in nucleic acid molecules, and can often shrink tumors in a matter of days after intravenous administration.
• 2-chloroethyl- nitrosoureas such as bis(2-chloroethyl)nitrosourea (BCNU), mitomycin, cyclophosphamide (cytoxan), and ifosphamide.
• BCNU bis(2-chloroethyl)nitrosourea
• cytoxan cyclophosphamide
• ifosphamide ifosphamide
• bistriazene compounds of the instant invention as chemotherapeutic and crosslinking agents has yet to be reported.
• the bistriazene compounds of the present invention are novel alkylating agents which are structurally similar to polyamines such as spermine and spermidine, which interact with DNA. Most currently employed chemotherapeutic alkylating agents interact covalently or noncovalently with the target DNA, after which a crosslinking reaction may occur.
• the bistriazene compounds of the present invention differ from any known chemotherapeutic agents in that their chemical structure allows them to interact with the DNA molecule while maintaining their chemical integrity. This interaction depends on the formation of multiple hydrogen bonds with the DNA, and in this manner they appear to mimic natural polyamines which normally interact with DNA.
• the bistriazenes may occupy the same sites in DNA as the polyamines themselves. Subsequent to this binding, the bistriazenes decompose on the surface of the DNA, releasing the "Linker" in the form of a bisdiazonium ion. This highly reactive substance covalently interacts with the DNA, causing multiple double strand breaks and interstrand crosslinks. As the bisdiazonium ion can be made to vary in its properties by structural modification of the Linker in the bistriazene molecule, the reactivity of the entire molecule can be modulated by appropriate chemical modification.
• bistriazenes may interact with DNA in a polyamine-like fashion, subsequently breaking down to form crosslinking agents which result in the formation of crosslinks lethal to cells.
• the use of the bistriazene compounds of the present invention as chemotherapeutic drugs therefore confers great specificity of drug interaction with DNA, and because the reactive diazonium ions are formed on the surface of the DNA, delivers a much higher effective dose of the ultimate cytotoxic agent per molecule of administered compound than for simple monodentate drugs.
• This feature achieves the advantage that the dosage of such bistriazene-based drugs administered will be low in comparison to that of other conventional chemotherapeutic alkylating agents.
• the combination of specificity and low effective dose portends bistriazene-based anti-cancer drugs with much lower systemic toxicities than those currently in use.
• the bistriazene compounds of the present invention represent an entirely novel class of bidentate, chemotherapeutic alkylating agents with greater specificity and lower toxicicity as compared to present treatments.
• a bistriazene compound such as bis (methyltriazeno)-p- xylene, bis (methyltriazeno)-2-butene, bis (methyltriazeno) ethane, and other bistriazene derivatives.
• Another object of the present invention is to provide a method for treating cancer in a mammal, including humans, which comprises administering to the subject an anti-cancer effective amount of a bistriazene compound and an anti-cancer effective amount of a an alkylating agent such as chlorambucil, melphalan, uracil mustard NF, cyclophosphamide, mechlorethamine hydrochloride, carmustine (BCNU), lomustine, dacarbazine
• a an alkylating agent such as chlorambucil, melphalan, uracil mustard NF, cyclophosphamide, mechlorethamine hydrochloride, carmustine (BCNU), lomustine, dacarbazine
• a further object of the present invention is to provide a pharmaceutical composition comprising an anticancer effective amount of a bistriazene compound.
• Yet a further object of the present invention is to provide a pharmaceutical composition
• a pharmaceutical composition comprising an anti-cancer effective amount of a bistriazene compound or an anti-cancer effective amount of bistriazene compound and an anti-cancer effective amount of an alkylating agent such as chlorambucil, melphalan, uracil mustard NF, cyclophosphamide, mechlorethamine hydrochloride, carmustine (BCNU), lomustine, dacarbazine (DTIC), thiotepa NF, and busulfan, or combinations of these latter compounds.
• an alkylating agent such as chlorambucil, melphalan, uracil mustard NF, cyclophosphamide, mechlorethamine hydrochloride, carmustine (BCNU), lomustine, dacarbazine (DTIC), thiotepa NF, and busulfan, or combinations of these latter compounds.
• bistriazene compounds useful in treating cancer include bis (methyltriazeno)-p-xylene, bis (methyltriazeno)-2- butene, bis(methyltri- azeno) ethane, and other bistriazene derivatives which are anti-cancer agents, such as the following:
• the compounds of the present invention can be used for the treatment of human and animal cancers.
• bistriazene compounds for the therapeutic treatment of neoplastic disease
• these compounds are also envisioned as another object of the present invention.
• reagents are often employed which interact with the molecule of interest such that the molecule is:
• toxins e.g., toxins to antibodies, antibodies to enzymes, small molecules to oligonucleotide DNA probes, etc.
• Bistriazenes can be adapted for use in these and other laboratory manipulations of macromolecules.
• bistriazene is modified such that the substituent groups afford a high degree of sequence recognition, then upon alkylation at a labile site, breakage of the DNA or protein backbone may occur (#1 above). Alternatively, alkylation at a stable site may block enzymatic digestion such as restriction enzyme digestion of DNA or protease digestion of proteins (#2 above). Multifunctional chemical crosslinking agents are presently widely used in applications #3, #5, and #6 cited above. The use of bistriazene compounds in such applications is another object of the present invention.
• bistriazene compounds will be employed as highly active chemical crosslinking agents useful in immobilizing molecules such as RNA, DNA or proteins on nitrocellulose, nylon, or other similar membranes (application #4, above), thereby facilitating the handling and probing of these biopolymers on such membrane supports.
• Yet another object of the present invention is to employ bistriazenes as crosslinking agents in the formation of chemical polymers from their monomeric constituents.
• bistriazene compounds of the present invention may be employed as chemical crosslinking agents in a manner similar to that of other well known crosslinking agents, as would be apparent to one of ordinary skill in the art.
• Figure 1 shows the survival in vitro of several human tumor cell lines exposed to various concentrations of bis (methyltriazeno)-p-xylene.
• Figure 2 shows the survival in vitro of several human tumor cell lines exposed to various concentrations of bis (methyltriazeno)-2-butene.
• Figure 3 shows the survival in vitro of several human tumor cell lines exposed to various concentration of bis (methyltriazeno) ethane.
• FIG 4 shows the survival in vitro of several human tumor cell lines exposed to various concentrations of 5- (3,3-dimethyltriazeno) imidazole-4-carboxamide (DTIC).
• CXF Colon Cancer Xenograft
• GXF Gastric
• LXF Lung: A adeno, L large cell, E epidermoid cell, S small cell
• MAXF Mammary Cancer Xenograft
• MEXF Melanoma PXF,
• Pleuramesothelioma SXF, Sarcoma; TXF, Testi ⁇ ular; XF, miscellaneous Cancer Xenograft.
• Figure 5 shows the results of the oligonucleotide crosslinking assay.
• Figure 6 shows the results of the supercoiled plasmid DNA assay.
• the basic bistriazene structure contains a number of elements which can be modified to affect the desired use of these compounds. These elements are indicated in the following structure:
• Linker moiety is involved in the structural definition of the molecule and in crosslink formation.
• the Linker can be either an alkyl group, substituted alkyl (including, but not limited to, alkylamines, alkyl ethers and thioethers, haloalkyl, silanes, phosphines, alcohols, amines, etc.), of chain length 1-20, preferably 2-8.
• the Linker may also include aralkyl or substituted aralkyl (with modifications analogous to those for substituted alkyls), polycyclic aralkyl, heterocyclic aralkyl, and their substituted derivatives wherein the triazine moieties can be separated by 1-30 carbon atoms, preferably 4-12 carbon atoms.
• aralkyl or substituted aralkyl with modifications analogous to those for substituted alkyls
• polycyclic aralkyl polycyclic aralkyl
• heterocyclic aralkyl and their substituted derivatives wherein the triazine moieties can be separated by 1-30 carbon atoms, preferably 4-12 carbon atoms.
• EG End Group
• the EGs may be identical or independently selected from groups comprising alkyl groups, substituted alkyl (including, but not limited to, alkylamines, alkyl ethers and thioethers, haloalkyl, silanes, phosphines, alcohols, amines, etc.), of chain length 1-20, preferably 1-6.
• the EG may also include aralkyl or substituted aralkyl (with modifications analogous to those for substituted alkyls), polycyclic aralkyl, aryl groups and heterocyclic groups of 2-40 non-hydrogen atoms, containing 1-6 rings, including nucleic acid bases and oligonucleotides.
• R or R' The final substituent on the triazene moiety, i.e., R or R', is perhaps the most fungible, and may be added following assembly of the basic triazene moiety by methods described for simple dialkyltriazenes (R.H. Smith, Jr., et al . , J. Org. Chem. , 1986 , 51 , 3751 ; R. H. Smith, Jr. , et al., J. Org. Chem. , 1988, 53 , 1467; D.H. Sieh, et al., J. Am. Chem. Soc . , 1980, 202, 3883; R.H. Smith, Jr. and CJ.
• R or R' may be identical to EG or to one another, or may be independently selected from the groups comprising hydrogen, alkyl groups, substituted alkyl (including, but not limited to, alkylamines, alkyl ethers and thioethers, haloalkyl, silanes, phosphines, alcohols, amines, etc.) of chain length 1-20, preferably 1-6.
• R or R' may also include aralkyl or substituted aralkyl (with modifications analogous to those for substituted alkyls), polycyclic aralkyl, aryl groups, and heterocyclic groups of 2-40 non-hydrogen atoms, containing 1-6 rings.
• R may be an acid derivative where the original acid includes, but is not limited to, carboxylic, sulfuric, sulfonic, phosphoric, phosphinic, arsenic, and selenic acids.
• R may also include, in the examples cited above, compounds where R equals R', or R is linked to R' such that a cyclic bistriazene compound is formed.
• Cases where R equals R' may be expanded to include multivalent metals including, but not limited to, palladium, platinum, titanium, zirconium, silicon, selenium, magnesium, and copper.
• metal species such as cisplatin and titanocene dichloride are clinically active as antineoplastic agents, and the bistriazene moiety may serve as a bidentate ligand for these classes of compounds in order to generate compounds with multiple modes of cytotoxic action.
• R is linked to R', polymeric compounds may result in addition to cyclic bistriazenes.
• the polymers produced would have unusual physical properties due to the hydrolytic instability of triazenes. It can be envisioned that this can be used to prepare polymers which could be implanted, and which would hydrolytically decompose to produce active cytotoxic agent in a time release manner. Similarly, it may be that the polymer would only provide a slowly dissolving matrix. This matrix may be used for structural applications, or to release an entrapped substance.
• bistriazenes are prepared by the reaction of 1,w-diazidoalkanes with two equivalents of an alkyllithium.
• the diazidoalkanes are prepared from the corresponding dihaloalkanes and sodium azide in dimethylformamide solution.
• BMTE 1,2-bis (methyltriazeno) ethane
• BMTE 1,2-bis (methyltriazeno) ethane
• BMTE is remarkably similar to polyamines such as spermine, spermidine, and their phosphatidyl derivatives, which are known to bind strongly to DNA.
• bistriazenes The synthesis and X-ray crystal structure of bistriazenes are described in Blumenstein et al., Tetrahedron Letters. submitted for publication, and Blumenstein et al., Chemical Communications, submitted for publication, respectively.
• the synthesis of particular bistriazenes is as follows:
• a flask is charged with 3.0 g (6.6 mmole) of trans-1,4-di(methyl 4-toluenesulfonate) cyclohexane, 1.08 g (16.6 mmole) of sodium azide, and 50 ml of dimethylformamide (DMF).
• the mixture is heated at 50°C with stirring under argon for 2 days.
• the mixture is then diluted with 150 ml of water and extracted four times with 40 ml of petroleum ether.
• the combined organic layers are dried over Na 2 SO 4 , filtered, and evaporated to afford a pale yellow oil.
• the residual oil is dissolved in 100 ml of anhydrous ether and cooled to - 20°C under argon.
• the bistriazene compounds of the present invention are useful in the treatment of a wide variety of cancers, as shown from the data below.
• Clonogenic Assay The response of a variety of human tumor cell lines to bistriazenes was determined via the clonogenic assay described in Fiebig et al. (1987) European Journal of Cancer and Clinical Oncology 23: 937- 948.
• the assay system consists of a modified, two-layer soft agar culture system.
• the bottom layer consists of 1 ml of modified Dulbecco medium supplemented with L-glutamine, containing 10% fetal calf serum and 0.5% agar, in a 35 mm petri-dish.
• the upper layer contains 2-5 x 10 5 viable human tumor cells suspended in a 1 ml volume, consisting of 0.3% agar, 30% fetal calf serum, and the medium.
• the drugs to be tested contained in 1 ml of medium containing 30% fetal calf serum, are included in the upper layer.
• Control plates are identical, except for the omission of the drugs. The plates are incubated at 37°C in a humidified atmosphere containing 7% carbon dioxide for varying periods (7-21 days). The time in culture is determined by the rate of colony formation in the control plates.
• the number of colonies in the drug treated cultures is compared to the number of colonies in the control plates, after visualization of the live colonies by staining with tetrazolium chloride.
• the tumors included those derived from colon cancer, three types of lung cancer, mammary cancer, ovarian cancer, two types of kidney cancer, a mesothelioma, a gastric cancer, and a sarcoma. These tumors represent some of the most important cancers for which current treatments are inadequate.
• the assays of the various bistriazenes were compared to the response induced in the same tumors by DTIC, a drug employed in clinical practice.
• Figure 1 shows dose-response curves obtained in the in vitro clonogenic cytotoxicity assay against several human tumor cell lines employing bis (methyltriazeno)-p-xylene. At a dose of 100 ug/ml, this compound was highly toxic to all tumor cell lines. At a dose of 10 ug/ml, it exhibited toxicity against approximately half of the cell lines examined. Some activity was also evident at a dose of 1 ug/ml in about half the cell lines.
• Figure 3 discloses the results obtained with bis (methyltriazeno) ethane in the clonogenic assay. This compound was highly cytotoxic at 100 ug/ml to most of the tumor cell lines. Relatively little or no activity was observed, however, in the mesothelioma, the gastric carcinoma, or the renal cancer RXF 423/17. At 10 ug/ml, only marginal, but significant, activity was seen in the large cell lung cancer and in the mammary cancer.
• DTIC (5-(3,3-dimethyltriazeno)imidazole-4-carboxamide) was tested in these cell lines. The results are shown in Figure 4. DTIC is used clinically against metastatic melanoma, non-Hodgkins lymphoma, and soft-tissue sarcomas. At each point, the dose of DTIC was 3 times larger than that of the bistriazenes. Thus, at 300 ug/ml, DTIC was potently cytotoxic on all cell lines. At 30 ug/ml, it showed activity against the gastric carcinoma GXF251/16 and the ovarian cancer OVXF899/9. At 3 ug/ml, it exhibited marginal activity against the gastric cancer.
• bistriazene 1,4-bis(methyltriazeno)-trans-2-butene is highly potent against several tumors, especially the large cell lung carcinoma.
• bistriazenes as a class of compounds, are cytotoxic agents which exhibit considerable selectivity toward certain tumors. It is also clear from these data that the nature of the Linker is of paramount importance in modulating the activity and selectivity of cytotoxic action of these compounds.
• the clonogenic assay system facilitates rapid testing of the anti-tumor activities of newly sythesized bistriazenes containing systematically varied EG's and Linkers, in order to establish the chemical and biological characteristics which will result in additional useful drugs.
• Bistriazenes react with varying efficiency with different oligonucleotides.
• Unsaturated bistriazenes such as p-xylyl and trans-butenyl produce stable crosslinked species in oligonucleotides.
• the amount of crosslinked species varies with the oligonucleotide sequence.
• the level of crosslinking is comprable to that seen with nitrogen mustard, and exceeds that observed with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.
• Final concentrations of the compounds in the oligonucleotide solution were 0.1 mM NMUST, 1.0 mM CCNU, or 10 mM bistriazene. Reactions were incubated at 37°C for 42 hours, and analyzed by denaturing polyacrylamide gel electrophoresis (20% gel) followed by autoradiography. the gels were intentionally overexposed to better visualize bands corresponding to interstrand crosslinks. Large amounts of unreacted oligonucleotides were also visualized under these conditions.
• Plasmid DNA Strandbreaking may occur via the hydrolysis of labile alkylation sites. A single strand break allows the relaxtion of supercoiled DNA to afford a nicked open circular form. Double strand breakage producing linear plasmid DNA occurs upon the hydrolysis of two labile alkylation sites close to one another on opposite DNA strands. These alkylation events may be either an interstrand crosslink, or discrete, but closely located, monoalkylations.
• Dialkyltriazenes afford more strand breakage than alkylsulfates and sulfonates. Bistriazenes are approximately 10-200 times more efficacious at producing strand breaks than dialkyltriazenes. Bistriazenes afford significant quantities of linear DNA, whereas simple dialkyltriazenes produce only small amounts of the linear form, and only traces are detectable in the reaction of alkylsulfates with plasmid DNA. Restriction endonuclease treatment of bistriazene-modified DNA suggests that linearization is not highly specific for sequences on the plasmid.
• the supercoiled plasmid strand break assay was carried out in a solution of 0.15 ug of pBR322 DNA in 9.5 ul of TE buffer (10 mM) Tris, 0.1, mM EDTA, pH 7.4) prepared at room temperature. A 0.5 ul aliquot of compound in DMSO was added, the solution vortexed lightly, and the samples incubated at 37° for 48 hours. Loading buffer (2 ul, 40% glycerol, and 1% bromphenol blue in TAE buffer) was added to each sample, and a 3 ul aliquot was analyzed by agarose gel electrophoresis (0.9% gel, 1.5 ug ethidium bromide/ml gel), and visualized by fluorescence.
• the bistriazene compounds of the present invention can be formulated into a pharmaceutical composition comprising an effective anti-cancer amount of the compound and a pharmaceutically acceptable carrier.
• An effective anti-cancer amount of the pharmaceutical composition will be administered to the subject, human, animal, or mammal, in a manner which inhibits cancer cell growth or replication.
• the amount of the compound and the specific pharmaceutically acceptable carrier will vary depending upon the host and its condition, the mode of administration, and the type of cancer being treated.
• the pharmaceutical composition comprises a bistriazene anti-cancer compound in effective unit dosage form.
• a bistriazene anti-cancer compound in effective unit dosage form.
• an effective unit dosage or “effective unit dose” is denoted to mean a predetermined anti-cancer amount sufficient to be effective against the cancer in vivo.
• Pharmaceutically acceptable carriers are materials useful for the purpose of administering the medicament, which are preferably non-toxic, and may be liquid materials which are otherwise inert and medically acceptable, and are compatible with the active ingredients.
• the pharmaceutical compositions may contain other active ingredients such as antimicrobial agents and other agents such as preservatives.
• compositions may take the form of a solution, an emulsion, suspension, ointment or cream. They may be administered parenterally, orally or topically, as an aerosol, spray, or drops, said parenteral administration being conducted intraperitoneally, intramuscularly, subcutaneously, intravenously, intraarticularly, intraarterially, or transdermally, depending upon whether the preparation is used to treat internal or external cancers.
• compositions may contain the compound in an amount of from about 0.1 % - about 99% by weight of the total composition, preferably about 1 to about 90% by weight of the total composition.
• the bistriazene compound can be dissolved in a pharmaceutically suitable carrier such as purified corn oil, propylene glycol, triolene, or dimethyl sulfoxide, and the dose may be about 0. 1 mg to about 1000 mg per kilogram per day.
• the compounds may be dissolved in a suitable vehicle, as above, and the dose may be about 1 mg to about 500 mg per kilogram per day.
• the compounds can be dissolved in oil or another compatible vehicle, and the dose can be about 0.1 mg to about 1000 mg per kilogram per day.
• injections can be carried out once or several times per day over a five day course depending upon the route of administration and the condition of the patient. After such courses, a recovery period of various length may be necessary. Additional courses may then be required under specific conditions.
• Total adult doses can range from about 0.1 to about 5000 mg, with dosages in the range of from about 10 to about 1000 mg being preferred.
• oral administration of bistriazenes encapsulated in liposomes or time-release formulations or dispersed in compatible emulsions together with stabilizing and/or dispersing agents may be the method of choice.
• bistriazenes may be formulated in oil or cream.

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